Nitrogen deposition reshapes biological nitrogen fixation and soil diazotrophic communities across urban and rural forests

Urban expansion on a global scale exposes the semi-natural environment to higher nitrogen (N) deposition disturbances with multiple uncertain consequences. One uncertainty is how N inputs impact biological N fixation (BNF), a crucial process supporting the entrance of N into terrestrial ecosystems through living organisms. Based on 5-year field experiments with different levels of N addition (0, 50 and 100 kg N ha⁻¹ yr⁻¹) in forests along an urban–rural gradient, we systematically examined the direct and indirect effects of N addition on soil BNF and its potential mechanisms. We found that soil BNF activity was drastically reduced (dropped by 94.2 % to 96.5 %) in both urban and suburban forests compared with the rural forest without additional N application. Nitrogen addition treatments had no effect on BNF activity in the urban forest, but significantly decreased BNF activity in the rural forest by over 50 % with low N addition. Further analysis revealed that reductions in BNF activity were associated with changes in the composition of diazotrophic communities, favoring facultative diazotrophs that are detrimental to soil BNF. Soil acidification was primarily responsible for limiting soil BNF and associated microbes in the urban forest. These findings indicate that external N inputs primarily pose a threat to soil diazotrophic communities and their N fixation function in the rural forest, but this negative effect does not persist in urban forests. Given the insensitivity of urban forests to external N inputs, it is essential to integrate human activity and land use contexts into soil behavior and N cycling predictions to better understand global N deposition effects.